Abstract:

The present invention relates in general to compositions, processes and
apparatus for imaging, and in particular for improved preparation,
collection and processing of images of specimens that include cartilage,
particularly specimens of intact or disarticulated joints. Images of
specimens according to the present invention include images obtained from
X-ray microscopic computed tomography.

Claims:

1. A method of producing a microCT image of a first stained specimen,
wherein said specimen comprises cartilage, said method comprising:(a)
incubating said specimen in a first staining composition wherein said
first staining composition comprises a 1% PTA solution, to produce a
first stained specimen; and(b) scanning said first stained specimen in an
X-ray tomography scanner,thereby producing said microCT image of said
first stained specimen.

2. The method of claim 1, wherein said specimen is a dissected knee joint.

3. The method of claim 1, wherein prior to said incubating step (a), said
specimen is placed in a fixative comprising formalin.

4. The method of claim 1, wherein said first staining composition further
comprises calcium, phosphate, or both calcium and phosphate,

5. The method of claim 1, said method further comprising:(a) incubating
said first stained specimen in a second staining composition, wherein
said second staining composition comprises a 5% PTA solution, to produce
a second stained specimen; and(b) scanning said second stained specimen
in an X-ray tomography scanner to produce a microCT image of said second
stained specimen.

6. The method of claim 5, wherein said microCT image of said first stained
specimen and said microCT image of said second stained specimen are
processed to identify anatomical features present in said image of said
second stained specimen that are not present in said image of said first
stained specimen.

7. The method of claim 5, wherein said second staining composition further
comprises calcium, phosphate, or both calcium and phosphate.

8. The method of claim 1, wherein prior to said scanning step (b), said
first stained specimen is injected with an injectable component.

10. A method of producing a microCT image of an intact joint, said method
comprising:(a) incubating an intact joint in a contrast agent to produce
a stained intact joint;(b) scanning said stained intact joint in an X-ray
tomography scanner,thereby producing said microCT image of said stained
intact joint.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001]This application claims the benefit of priority of U.S. Provisional
Patent Application Nos. 61/143,383, filed Jan. 8, 2009, and 61/186,975,
filed Jun. 15, 2009, each of which is hereby incorporated by reference in
its entirety.

FIELD OF THE INVENTION

[0002]The present invention relates in general to compositions, processes
and apparatus for imaging, and in particular for preparation, collection
and processing of specimens containing cartilage to produce high
resolution images, including images obtained with X-ray microscopic
computed tomography.

BACKGROUND OF THE INVENTION

[0003]Obtaining high resolution images of joints, particularly the
differentiation between bone and cartilage, can be of benefit for the
diagnosis and treatment of disorders and diseases that affect joints and
other regions of the body that are prone to inflammation of cartilage and
other connective tissue. However, high resolution images of the boundary
region between bone and cartilage can be difficult to obtain using
standard microcomputed tomography ("microCT") imaging techniques.

[0004]Conventional bone and tissues analyses are performed by
ashing/caliper measures and tissue staining on slides respectively. While
ashing and caliper bone quantitation have been replaced by microCT for
humans and larger animals, progress on small animal cartilage imaging has
been slower to develop, particularly since traditional histological
chemical staining is readily available and relatively inexpensive.
Imaging of smaller animals is of use in studies that require analysis of
large numbers of animal models, such as drug development and
environmental impact studies.

[0005]MicroCT-based virtual histology imaging provides a high resolution
system that can be simple to implement, relatively inexpensive, and more
rapid than comparable methods of phenotyping anatomy, particularly
anatomy of tissue samples, whole organs and whole organisms. Methods for
increasing resolution of images obtained from microCT-based methods would
be of benefit to applications utilizing such imaging methods.

SUMMARY OF THE INVENTION

[0006]Accordingly, the present invention provides methods and compositions
for increasing the resolution of images obtained from specimens,
particularly specimens comprising cartilage and bone. The resolution of
images obtained through methods such as microCT is increased by using
methods and compositions of the invention for obtaining, staining and
further processing specimens for imaging modalities.

[0007]In one aspect, the invention provides methods for producing a
microCT image of a specimen containing cartilage. In one embodiment, the
specimen is an intact or a disarticulated joint. In a further embodiment,
the joint is a knee joint. In a further aspect, the specimen is stained
using one or more staining compositions.

[0008]The advantage of the staining compositions and methods of the
present invention is that these methods allow measurement of cartilage
and the boundary between cartilage and bone. In traditional methods,
cartilage in preclinical specimens is essentially invisible to the
microCT scanner (see for example, FIG. 6, which is a microCT image of a
knee joint imaged in the absence of contrast agent). The present
invention allows visualization of soft tissues in joints (particularly
intact joints) at an index of refraction less than that of bone with a
refraction signature unique to cartilage by using radio-opaque contrast
agents that infuse and bind to the tissues themselves.

[0009]Traditional stains (such as haematoxylin and eosin, alcian blue,
alizarin red, Gomori's trichrome, and the like) only apply to stained 2-D
histological sections on glass slides and are not useful in CT because
X-rays are "blind" to these stains--i.e., these stains are radiolucent.
In addition, physical characteristics of the traditional stains make them
incompatible with many of the imaging methods described herein, such as
incomplete diffusion, toxicity to personnel in high volume, high
osmolarity gradients, and other disadvantages that are known in the art.

[0010]The methods and compositions described herein make cartilage
apparent to microCT, allowing resolution of the tissues in three
dimensions without resorting to damaging the specimen by slicing it into
thin pieces as is required in traditional methods. The present invention
allows one to digitally observe the specimen in all three planes, whereas
in traditional methods in which the cartilage cannot be resolved in the
image, an anatomical plane of interest must be chosen before sectioning
and the other two viewable planes are therefore lost thereafter for that
particular specimen.

[0011]In one aspect, staining compositions of the invention include PTA.
In an exemplary embodiment, staining compositions include a 1% PTA
solution. In a further embodiment, staining compositions also include
additives. In a still further embodiment, staining compositions include
one or more buffers.

[0012]In one aspect, staining compositions of the invention include an
iodinated contrast agent. In an exemplary embodiment, the iodinated
contrast agent includes ioxaglate.

[0013]In further embodiments, the specimen is stained multiple times prior
to imaging.

[0014]In some embodiments, subsequent to staining, the stained specimen is
injected with one or more compositions to provide increased resolution
between apposing cartilage plateaus. This further injection is conducted
prior to imaging.

[0015]In specific aspects, the stained specimen is scanned in an X-ray
tomography scanner to produce a microCT image.

[0016]Advantages of using microCT analysis of specimens containing
cartilage, particularly specimens that are the intact joints of small
animals (such as rodent models of disease) over traditional histology
include: the availability of three-dimensional images, flexible sample
orientation, rapid processing, high-resolution and the non-destruction of
specimens during imaging. In addition, bone and cartilage data for an
individual specimen can be analyzed using data and image processing
techniques, such as those described in U.S. application Ser. Nos.
12/162,376, filed Oct. 15, 2008 and 11/839,414, filed Aug. 15, 2007, each
of which is hereby incorporated by reference in its entirety for all
purposes and in particular for all teachings (including written
description, figures and examples) related to obtaining and analyzing
images obtained using microCT virtual histology methods. A further
advantage of methods of the present invention is that specimen integrity
is preserved during imaging, allowing for additional processing if
necessary.

BRIEF DESCRIPTION OF THE FIGURES

[0017]FIG. 1 is a microCT image from a specimen stained in a 1% PTA
solution containing calcium for four days.

[0018]FIG. 2 is a microCT image from a specimen stained in a 5% PTA
solution for three days.

[0020]FIG. 4 is a sagittal microCT image of an intact rat knee in which
the specimen was stained in Hexabrix and then injected with calcium
carbonate.

[0021]FIG. 5 is a coronal microCT image of an intact rat knee in which the
specimen was stained in Hexabrix and then injected with calcium
carbonate.

[0022]FIG. 6 is a microCT image of an intact rat knee where no contrast
agent was applied to the specimen.

[0023]FIG. 7 is a photograph of an exemplary intact knee joint specimen.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024]Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. All publications
mentioned herein are incorporated herein by reference for the purpose of
describing and disclosing devices, formulations and methodologies which
are described in the publication and which might be used in connection
with the presently described invention.

[0025]Where a range of values is provided, it is understood that each
intervening value, between the upper and lower limit of that range and
any other stated or intervening value in that stated range is encompassed
within the invention. The upper and lower limits of these smaller ranges
may independently be included in the smaller ranges, and are also
encompassed within the invention, subject to any specifically excluded
limit in the stated range. Where the stated range includes one or both of
the limits, ranges excluding either both of those included limits are
also included in the invention.

[0026]In the following description, numerous specific details are set
forth to provide a more thorough understanding of the present invention.
However, it will be apparent to one of skill in the art that the present
invention may be practiced without one or more of these specific details.
In other instances, well-known features and procedures well known to
those skilled in the art have not been described in order to avoid
obscuring the invention. It will be apparent to one of skill in the art
that these additional features are also encompassed by the present
invention.

DEFINITIONS

[0027]As used herein and in the appended claims, the singular forms "a,"
"an," and "the" include plural referents unless the context clearly
dictates otherwise. Thus, for example, reference to "an agent" refers to
one agent or mixtures of such agents, and reference to "the method"
includes reference to equivalent steps and methods known to those skilled
in the art, and so forth.

[0028]Unless defined otherwise, all technical and scientific terms used
herein generally have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Generally, the
nomenclature used herein and the laboratory procedures in cell culture,
molecular genetics, organic chemistry, and nucleic acid chemistry and
hybridization described below are those well known and commonly employed
in the art. Standard techniques are used for nucleic acid and peptide
synthesis. The techniques and procedures are generally performed
according to conventional methods in the art and various general
references (see generally, Sambrook et al. MOLECULAR CLONING: A
LABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., which is incorporated herein by reference),
which are provided throughout this document. The nomenclature used herein
and the laboratory procedures in analytical chemistry, and organic
synthetic described below are those well known and commonly employed in
the art. Standard techniques, or modifications thereof, are used for
chemical syntheses and chemical analyses.

[0029]As used herein, a "specimen" is a biological specimen, which
encompasses cells, tissues (including bone and joints), organs and whole
organisms. The term "specimen" is used interchangeably with the term
"sample" herein.

[0030]As used herein, the term "organism" refers to any living entity
comprised of at least one cell. A living organism can be as simple as,
for example, a single eukaryotic cell or as complex as a mammal. The term
"organism" encompasses naturally occurring as well as synthetic entities
produced through a bioengineering method such as genetic engineering.

[0031]As used herein, the term "tissue" includes cells, tissues, organs,
blood and plasma.

[0032]The term "identifying" (as in "identifying an anatomical feature")
refers to methods of analyzing an object or property, and is meant to
include detecting, measuring, analyzing and screening for that object or
property.

[0033]A "property" is any biological feature that can be detected and
measured.

[0034]The term "diagnosing disease" encompasses detecting the presence of
disease, determining the risk of contracting the disease, monitoring the
progress and determining the stage of the disease.

[0035]The "determining effectiveness of a treatment" includes both
qualitative and quantitative analysis of effects of a treatment.
Determining effectiveness of a treatment can be accomplished using in
vitro and/or in vivo method. Determining effectiveness of a treatment can
also be accomplished in a patient receiving the treatment or in a model
system of the disease to which the treatment has been applied. In
general, determining effectiveness of a treatment includes measuring a
biological property at serial time points before, during and after
treatment to evaluate the effects of the treatment.

[0036]"Treatment" generally refers to a therapeutic application intended
to alleviate, mitigate or cure a disease or illness. Treatment may also
be a therapeutic intervention meant to improve health or physiology, or
to have some other effect on health, physiology and/or biological state.
Treatment includes pharmacological intervention, radiation therapy,
chemotherapy, transplantation of tissue (including cells, organs, and
blood), and any other application intended to affect biological or
pathological conditions.

[0037]The term "subject" refers to an organism that is the recipient of a
biological and/or therapeutic intervention. A subject can be any
organism, including cells, animals, and plants.

[0038]The term "patient" refers to a human subject that has a disease or
has the potential of contracting a disease.

[0040]The term "virtual histology" refers to methods by which specific
tissues can be visualized using stains of the invention.

Overview

[0041]The present invention provides compositions and methods for imaging
specimens. In particular, the present invention provides compositions and
methods for preparing specimens for imaging modalities (such as microCT
virtual histology) to obtain images of cartilage, particularly cartilage
in and around joints.

[0042]In one aspect, the present invention provides staining compositions
for preparing specimens for imaging. Staining compositions of the
invention are in specific embodiments tailored to improve the resolution
of images obtained from specimens that include cartilage and bone, such
as intact and disarticulated joints.

[0043]In further aspects, stains of the invention include electron dense
staining agents such as phosphotungstic acid and ioxaglate.

[0044]In further aspects, specimens are processed to further improve the
resolution provided by the stains. In some embodiments, incisions are
made in certain regions of the specimens to improve penetration of the
stains. In further embodiments, specimens are placed in fixatives prior
to staining. In still further embodiments, specimens are injected with
compositions to increase the contrast between apposing cartilage
structures in stained specimens. In yet further embodiments, additives
are included in the staining compositions that further improve the
contrast between anatomical features.

[0045]In a further aspect, the present invention provides methods for
obtaining images of specimens prepared according to the methods described
herein. Any imaging modality known in the art can be used in methods of
the present invention. In specific embodiments, imaging is accomplished
using microCT virtual histology methods.

[0046]The methods and compositions of the present invention can be used in
accordance with and/or in combination with the teachings of U.S.
application Ser. Nos. 12/162,376, filed Oct. 15, 2008; 11/575,057, filed
Jan. 29, 2008; 11/888,995, filed Aug. 3, 2007; 11/839,414, filed Aug. 15,
2007; 12/389,094, filed Feb. 19, 2009; 61/143,380, filed Jan. 8, 2009;
and 61/230,574, filed Jul. 31, 2009, each of which is hereby incorporated
by reference in its entirety, including all drawings, examples, and
disclosure.

Preparing Specimens for Imaging

[0047]In one aspect, the present invention provides methods and
compositions for preparing specimens for acquisition of images. Preparing
specimens for imaging includes dissection and further incisions upon the
dissected specimen, fixing the specimens in one or more fixatives,
staining the specimens in one or more staining agents that may include
one or more additives, and further introducing additional compositions to
improve the contrast between specific anatomical features.

[0048]In further embodiments, combinations of preparation methods are used
to process specimens for imaging. As will be appreciated, any combination
of such preparation methods described herein and known in the art can be
used in accordance with the present invention. In some embodiments,
staining agents are optionally combined with a buffer and/or a fixative
and/or a cross-linking agent and/or a reporter substrate for a reporter
gene product. As will be appreciated, any combination of such materials
can be used to stain specimens in accordance with the present invention.

[0049]Staining Compositions

[0050]In one aspect, the invention provides staining compositions for
preparing specimens for acquisition of images, such as microCT virtual
histology images. Some components of such staining compositions are known
in the art and described, for example, in International Publication No.
WO/2007/089641, filed on Jan. 26, 2007 and U.S. application Ser. No.
11/575,057, filed Oct. 23, 2008, each of which is hereby incorporated by
reference in its entirety for all purposes and in particular for all
teachings related to preparing specimens for imaging, particularly
microCT virtual histology imaging. In general, staining compositions of
the present invention include staining agents (also referred to herein as
"stains") and may also include additional components such as buffers,
fixatives, additives, and combinations of any of these. Staining
compositions of the present invention show an increased resolution over
conventional stains, particularly with respect to boundary
differentiation between bone and cartilage in visualization methods such
as microCT.

[0052]In some embodiments, staining agents of use in the invention are
iodinated contrast agents, such as ioxaglate (i.e., Hexabrix).

[0053]In general, the staining agent is present in staining compositions
of the invention in any concentration useful to provide a desired level
of contrast in the image of the specimen. Appropriate concentrations of a
selected staining agent are readily determinable by those of skill in the
art without resort to undue experimentation. For example, arrays of
staining compositions including a single staining agent are prepared.
Each composition is used to stain a specimen. The level of staining of
each specimen by each staining composition is determined by acquiring a
microCT virtual histology image of each of the stained specimens.

[0054]In an exemplary embodiment, the staining agent is present in the
staining composition in an amount from about 0.01 weight percent to about
10 weight percent, preferably from about 0.1 weight percent to about 5
weight percent, more preferably from about 1 weight percent to about 3
weight percent.

[0055]Optionally, staining compositions of the invention further include
at least one buffer component. The buffer is present in any concentration
that is useful to provide a desired level of staining of the specimen, as
evidenced, in one embodiment, by obtaining a desired level of contrast in
a microCT image of the stained tissue. A buffer that has a different
osmotic concentration than the tissue is optionally used in the process
of stain penetration so as to accelerate transfer of stain molecules into
components of the tissue, e.g., tissue cells.

[0056]Exemplary buffer concentrations for staining compositions of the
invention range from about 0.01 M to about 1.0 M. In further exemplary
embodiments, the buffer concentrations are in the range of about 0.05 to
about 0.90, about 0.10 to about 0.80, about 0.20 to about 0.70, about
0.30 to about 0.60 and about 0.40 to about 0.50 M. In some embodiments,
the buffer is a cacodylate buffer, e.g., sodium cacodylate trihydrate. In
some embodiments, the buffer is a phosphate buffer. Other buffers known
in the art may also be used in accordance with the present invention.

[0057]In further embodiments, staining compositions include at least one
fixative or cross-linking agent component such as glutaraldehyde,
formaldehyde, alcohols, or a combination of these. In exemplary staining
compositions, the fixative or cross-linking agent is present in a
concentration range of from about 0.05% to about 5%, preferably from
about 0.1% to about 3% and more preferably from about 1% to about 1.5%.

[0058]In still further embodiments, staining compositions of the invention
may also include a tissue penetration enhancing agent component. A
representative tissue penetration enhancing agent is DMSO.

[0059]In further embodiments, staining compositions of the invention
include both the staining agent and a species that is indicative or
confirmative of the presence of a reporter gene through direct
interaction with that gene or with a product of the reporter gene. In one
embodiment, the reporter gene product forms a complex with the species
recited above and the staining agent. The resulting agent is detectable
by an imaging modality, e.g., an X-ray imaging modality, such as microCT.

[0060]In yet further embodiments, staining compositions of the invention
may include at least one additive component. Such additives can be useful
for semi-automated computational analysis of the resultant images,
because these additives can help preserve bone landmarks (for example,
trabecular structures). Preservation of bone landmarks allows data sets
to be iteratively overlaid with accuracy. In specific embodiments, these
additives include aqueous calcium. In further embodiments, aqueous
calcium in the concentration of about 0.1 to about 5 M is used. In still
further embodiments, aqueous calcium in the concentration of about 0.2 to
about 4, about 0.3 to about 3, about 0.4 to about 2, and about 0.5 to
about 1 M is used in staining compositions of the invention. In further
specific embodiments, additives used in staining compositions of the
invention include without limitation: calcium, potassium, manganese,
magnesium, silica, iron, zinc, selenium, boron, phosphorus, sulfur,
chromium, hydroxyapatite. As will be appreciated, such additives can be
used individually or in combination with other additives or any of the
other components of staining compositions described herein.

[0061]In still further embodiments, any combination of the above
components is included in staining compositions of the present invention.

[0062]PTA Solutions

[0063]In specific embodiments, the staining agent used in staining
compositions of the invention is phosphotungstic acid (PTA). In further
embodiments, the PTA is present in concentrations from about 3 to about
10 weight percent PTA. In still further embodiments, the PTA is present
in concentrations in the range of about 4 to about 9, about 5 to about 8,
and about 6 to about 7 weight percent PTA. In yet further embodiments,
the PTA is present in a range from about 4.8 to about 5.2 weight percent
PTA.

[0064]In further embodiments, staining compositions of the invention
include PTA solutions in combination with calcium. In still further
embodiments, the PTA solutions include phosphate. The addition of calcium
and/or phosphate can include the resolution of images obtained using
imaging applications such as microCT. For example, FIG. 1 shows a
specimen stained for four days in a 1% PTA solution containing calcium.

[0065]In specific embodiments, a cartilage stain of the invention
comprises a 1% PTA solution with 0.8 mM calcium chloride and a 10×
phosphate buffer (10-fold dilution of a phosphate buffer comprising
Na2HPO4/KH2PO4 at pH 7.4). It will be appreciated
that the concentrations of the various components of the cartilage stain
can be varied and that such variations also fall within the scope of the
present invention. For example, the PTA solution may range from a 1% to a
20% solution. In yet further embodiments the PTA solution may range from
2%-18%, 3%-16%, 4%-14%, 5%-12%, 6%-10%, and 7%-8%. In a further example,
the calcium chloride concentration may range from about 0.5 mM to about
10.0 mM. In still further embodiments, the calcium chloride concentration
ranges from about 1.0 to about 9.0, about 1.5 to about 8.0, about 2.0 to
about 7.0, about 3.5 to about 6.5, about 4.0 to about 6.0, and about 4.5
to about 5.0 mM. In a still further example, the phosphate buffer may be
a 1×, 2×, 5×, or 10× solution.

[0066]It will be appreciated that various combinations of the above
described exemplary embodiments for staining compositions are encompassed
by the present invention, and that the components of the staining agents
described herein can be titrated to determine the combination that
produces an optimal image of a particular specimen. For example,
different PTA staining solutions will elucidate different anatomical
features of a specimen, as is evident when comparing the images in FIG. 1
and FIG. 2. FIG. 1 is a microCT image of a specimen stained in a 1% PTA
solution containing calcium for four days, and FIG. 2 is a microCT image
of a specimen stained in a 5% PTA solution for three days.

[0067]It will be further appreciated that PTA solutions can be used with
any of the other components of staining compositions described herein to
stain specimens. In specific embodiments, 1% PTA solutions are used in
combination with calcium and optionally other additives described herein
in staining compositions of the invention.

[0068]Iodinated Contrast Agents

[0069]In one aspect, the present invention provides contrast agents for
staining specimens that include cartilage, including specimens such as
joints (intact and disarticulated). In specific embodiments, the present
invention utilizes iodinated contrast agents such as ioxaglate i.e.,
Hexabrix (Mallinckrodt) to stain specimens for imaging.

[0070]In exemplary embodiments, staining compositions comprise
full-strength (i.e., undiluted) Hexabrix. In other embodiments, staining
compositions comprise a dilution of Hexabrix. In further embodiments, the
dilution may range from a 1:2 to a 1:100 dilution. The dilution of
Hexabrix may be in water, in a staining composition comprising any of the
components described herein, saline, or any other medium known in the
art.

[0071]It will be appreciated that iodinated contrast agents such as
Hexabrix can be used with any of the other components of staining
compositions described herein to stain specimens. In some embodiments,
Hexabrix is used in combination with calcium and optionally other
additives described herein in staining compositions of the invention.

[0072]Prior to incubation in a staining composition comprising Hexabrix,
the specimen may first be fixed using any of the methods and compositions
described herein and known in the art. In general, fixation prior to
Hexabrix staining is conducted in a 10% buffered formalin solution for at
least five days.

[0073]Subsequent to staining, the specimen may be injected with one or
more of the injectable components described herein and known in the art
to further delineate apposing cartilage layers.

[0074]Methods of Staining Specimens

[0075]Although staining agents are traditionally applied by oral
administration, intravenous administration or direct injection into the
area to be imaged, the present invention provides methods for staining
intact tissue by incubation in the agent. The present inventors have
found that although not traditionally thought to be able to penetrate
intact tissue, certain staining agents are able to pass through tissue of
an intact joint into the joint space to stain the specimen such that the
boundary between bone and soft tissue can be differentiated using
visualization methods such as microCT.

[0076]In an exemplary aspect, specimens are incubated for a selected
period in a staining composition of the present invention. The period of
time over which the specimen is incubated with the staining composition
is readily determined by those of skill in the art and is informed by the
level of contrast desired in the images acquired from the stained
specimen. Incubation in staining compositions is generally conducted at
ambient room temperature, but staining at higher and lower temperatures
is also within the scope of the present invention.

[0077]In exemplary embodiments, the specimen is in contact with the
staining compositions from about one hour to about one week. In still
further exemplary embodiments, the specimen is in contact with the
staining compositions for about nine hours to about five days, about
twelve hours to about four days, about sixteen hours to about two days
and about eighteen hours to about twenty-four hours. Periods of at least
about three hours, at least about five hours, at least about ten hours
and at least about fifteen hours are also of use in the methods of the
invention

[0078]In some embodiments, the specimen is serially stained with two or
more staining compositions. In further embodiments, such serial staining
is conducted using the same kinds of staining compositions or using
different kinds of staining compositions. For example, in some
embodiments, the preparation of a specimen for imaging of cartilage
comprises two separate PTA stains. In such embodiments, the specimen is
stained for a period of time in a first staining composition comprising a
PTA solution, and then re-stained in a second staining composition
comprising a PTA solution. The first and second staining compositions may
include identical PTA solutions or different PTA solutions. For example,
the first staining compositions may include a 1% PTA solution whereas the
second staining compositions may include a 1% PTA solution in combination
with an additive such as calcium. As will be appreciated, serially
staining as described herein can be conducted using staining compositions
with any combinations of components described herein and known in the
art.

[0079]In further embodiments, after incubation in a staining composition,
specimens are transferred to one or a series of buffer solutions so as to
remove extra staining agents and to create a density contrast between the
specimens and the bordering environment to facilitate distinguishing of
the tissue from its bordering environment. In some embodiments, the
buffer has a different osmolality than that of the tissue to accelerate
or otherwise enhance the transfer of stain molecules into components of
the specimen, e.g., tissue cells. An exemplary buffer is a buffered
saline solution, e.g., phosphate buffered saline (PBS). When this
subsequent osmolality differential is applied, the staining composition
can be of a greater or lesser osmolality than the buffer to which the
stained specimen is subsequently submitted. Buffer solutions of use in
the present invention can include without limitation sodium cacodylate
buffer, phosphate-buffered saline, and ethanol solutions. In specific
embodiments, transfers through buffers are conducted for the same or
different periods of time. In further embodiments, these transfers (also
referred to herein as "washes") through buffers are conducted for about
one to about five hours.

[0080]In yet further embodiments, the stained specimen may further be
submitted to treatment with an organic solvent or a mixture of an organic
solvent in water. Exemplary organic solvents are those that are at least
partially soluble in water and include, e.g., alcohols, ethers, esters
and the like. The medium in which the specimen is suspended can be
altered from a first mixture (e.g., the staining composition) to a final
mixture (e.g., 100% organic solvent) in a single step or, alternatively,
the change in specimen environment can be accomplished by submitting the
stained specimen to a gradient of medium compositions, moving step-wise
or continuously from the first mixture to the final mixture.

[0081]In some embodiments, specimens are fixed prior to contact with
staining compositions. In some embodiments, specimens are fixed through
incubation in a formalin solution for a period of time. In some
embodiments, the formalin is a 10% neutral buffered formalin solution. In
further embodiments, the formalin can range from a 0.5 to a 15% neutral
buffered solution. In some embodiments, the specimen is fixed for a
period of about two to four days. In further embodiments, the specimen is
fixed for a period of about one day to about two weeks. In still further
embodiments, the specimen may be fixed for a month or longer.

[0082]In still further embodiments, after contact with one or more
staining compositions, the stained specimen is further subjected to
additional injection of "injectable" components. Such injectable
components are of particular use in improving differentiation between
apposing cartilage layers, for example in the region of the synovial
space of a joint such as a knee joint. In exemplary embodiments, the
injectable components are radio-opaque injectables suspended in a medium
such as a lightweight oil or aqueous saline preparation. Other types of
medium are known in the art and can be used in accordance with the
present invention. Injectable components may also be suspended in a
composition of the same or similar makeup as the staining composition
used to stain the specimen. In specific embodiments, injectable
components used in accordance with the present invention include 50%
calcium carbonate suspended in a light oil medium. In further
embodiments, these injectable components include without limitation:
barium, barium sulfate, bismuth oxychloride, bismuth trioxide, bismuth
potassium tartrate, bismuth subcarbonate, bismuth sodium iodide, bismuth
sodium tartrate, bismuth sodium triglycollamate, bismuth subsalicylate,
bromine, calcium carbonate, calcium sulfate, calcium chloride, ferrous
carbonate, ferrous chloride, ferrous fumarate, ferrous gluconate, ferrous
iodide, ferrous lactate, ferrous sulfate, ferrous succinate, gold,
iodine, iron, magnesium oxide, magnesium sulfate, platinum, silver,
sodium carbonate, tungsten, zinc, zinc acetate, zinc carbonate, zinc
citrate, zinc iodate, zinc iodide, zinc lactate, zinc oxide, zinc
phosphate, zinc salicylate, zinc stearate, zinc sulfate, and all
combinations thereof. As will be appreciated, these injectable components
can be introduced into the sample, particularly the synovial space of
joints, using any method known in the art, including injection using a
syringe.

[0083]In further embodiments, specimens are washed prior to, subsequent
to, or both prior to and subsequent to incubation in a staining
composition. In still further embodiments, specimens are washed prior to,
subsequent to, or both prior to and subsequent to pre-stain fixation in
solutions such as formalin and/or prior to, subsequent to, or both prior
to and subsequent to exposure to injectables such as calcium carbonate in
light oil medium. In specific embodiments, these washes are conducted in
phosphate buffered saline (PBS) for about one to about five hours. In
still further embodiments, multiple washes are conducted.

[0084]The methods of the invention preferably provide stained specimens in
which the density of the staining is essentially invariant from one
border of the specimen to an antipodal border of the specimen. As used
herein, the term "essentially invariant" refers to the homogeneity of the
staining of a specimen. In a preferred embodiment, a specimen exhibiting
essentially invariant staining will have a density of stain that varies
by no more than about 20%, more preferably by no more than about 10% and
still more preferably by more than about 5% across a line through the
specimen from a point on one border of the specimen to the antipodal
point on the opposite border of the specimen.

[0085]As will be appreciated, any combination of methods and staining
compositions described herein can be used to prepare specimens for
imaging. Although specific embodiments of staining compositions and
methods are described herein, it is within the skill of one in the art to
alter components and procedures described herein and known in the art in
to prepare specimens for imaging modalities such a microCT virtual
histology.

[0086]In an exemplary embodiment, a specimen is prepared by first being
fixed in 10% neutral buffered formalin for about 4 to about 5 days. The
fixed specimen is then washed three times, one hour per wash, in PBS. The
washed specimen is then stained for four days in a staining composition
comprising a PTA solution. In further embodiments, the PTA solution is a
1% PTA solution. In still further embodiments, the 1% PTA solution will
include calcium and phosphate. In some embodiments, this staining is
conducted at room temperature. In further embodiments, the PTA solution
is exchanged for fresh solution each day. The specimen is again washed
three times, one hour per wash, in PBS and then subjected to a microCT
scan to produce a first image. The specimen is then re-stained in a
second PTA solution for three days. In some embodiments, the second PTA
solution is a 5% PTA solution. In further embodiments, the 5% PTA
solution will also include calcium and phosphate, as discussed above. In
further embodiments, the PTA solution is exchanged for fresh solution
each day. The re-stained specimen is then washed in PBS three times, one
hour for each wash, and then subjected to a microCT scan to produce a
second image. As will be appreciated, the types of PTA solutions, fixing
solutions, and the amount of time spent in each solution can be varied to
produce images of optimal resolution. In further exemplary embodiments,
the second image and the first image are processed using methods known in
the art to further elucidate anatomical features in the images. Such
methods are described for example in U.S. patent application Ser. Nos.
12/162,376, filed Oct. 15, 2008 and 11/839,414, filed Aug. 15, 2007, each
of which is hereby incorporated by reference in its entirety for all
purposes and in particular for all teachings, description, figures and
examples related to obtaining and processing images obtained using
microCT virtual histology methods. In some embodiments, processing of
multiple images obtained at various stages of staining involves a
subtraction procedure, which provides data regarding anatomical features
brought out by the re-staining process.

[0087]In a specific embodiment, an intact joint specimen is placed in an
iodinated contrast agent. In one non-limiting example, the iodinated
contrast agent is ioxaglate (Hexabrix). In a further embodiment, the
specimen in Hexabrix is placed on a rocker to allow the stain to
penetrate the specimen thoroughly. In some embodiments, the specimen is
incubated in the Hexabrix at ambient room temperature. As discussed
above, the specimen can be incubated in the Hexabrix for the amount of
time needed to achieve the best resolution for the visualization method
that is to be used. The staining may take place for about four hours to
about one week. In some further embodiments, the specimen is incubated in
the Hexabrix from about five to about forty-eight hours. In still further
embodiments, the specimen is incubated in the Hexabrix from about ten to
about thirty-six hours, from about twelve to about twenty-four hours, and
from about sixteen to about twenty hours. In yet further embodiments, the
specimen is incubated in the Hexabrix from about sixteen to about
twenty-four hours.

[0088]In further exemplary embodiments, after staining in Hexabrix, the
specimen is injected with an injectable. In still further embodiments,
the injectable is calcium carbonate. Injection of calcium carbonate
improves the differentiation between apposing cartilage layers (see for
example FIGS. 4 and 5, which show the saggital and coronal view of a rat
knee stained with a solution containing Hexabrix and then injected with
calcium carbonate suspended in Hexabrix).

[0089]The present inventors have found that although Hexabrix is of use in
staining intact joints, not all contrast agents provide equivalently high
quality images of intact (as opposed to disarticulated) joints. For
example, osmium tetroxide and ethidium bromide are highly toxic in
quantity, Ruthenium Red has low contrast, and Uranyl Acetate emits low
level radioactivity when used in staining intact joints.

[0090]Methods of Dissection and Further Preparation of Specimens for
Staining

[0091]In an exemplary aspect of the invention, the specimen stained is a
"solid tissue". As used herein, "solid tissue" refers to those tissues in
which the parenchyma is present in an amount of at least about 50%. Solid
tissue is distinct from tissue such as lung tissue.

[0092]In some embodiments, specimens stained according to the methods
described herein comprise joints, including knee joints. In further
embodiments, specimens such as knee joints are obtained from mammals such
as rats and mice using dissection methods known in the art and described
herein. In still further embodiments, specimens are prepared to enhance
the penetration of the stains using blanching methods, incisions, and
combinations of blanching and incisions.

[0093]Specimens of the invention can include joints, tissues, as well as
whole organisms, e.g, an embryo or fetus.

[0094]In a further embodiment, penetration of staining compositions into a
specimen is enhanced prior to or during treatment of the specimen with
the stain. In an exemplary method, the porosity of the specimen is
enhanced by chemical or physical methods. Exemplary chemical methods
include osmotic disruption of the integrity of the specimen structure and
treatment of the tissue with a penetration enhancing substance, e.g.,
DMSO. Physical means include, but are not limited to puncturing the
specimen to form channels in the specimen through which the stain flows
with greater facility than through corresponding undisrupted regions of
the specimen. Channels can be formed in the specimen by puncturing it
with an object or by subjecting it to focused energy, such as the light
from a laser. in a general example of a staining process of the
invention, a specimen, e.g., a cell, a tissue, an embryo, or a fetus, is
stained to saturation for a selected period in a solution of 0.1 M buffer
(pH 7.2), 1% fixative or cross-linking agent, and 1% staining agent,
rocking at room temperature. The stained specimen is then washed and
dehydrated. For example, specimens are washed for 30 minutes in 0.1M
buffer, and twice more for 30 minutes in a second buffer providing an
environment with an osmolality different from the staining solution
and/or the washing buffer subsequent to the staining solution. Specimens
are then incubated in a graded series of organic solvent concentrations
to 100% organic solvent prior to imaging. An organic solvent is an
example of a medium that increases the apparent density differences
between the suspension medium and the stained tissue. In an exemplary
staining process of the invention, a specimen, e.g., a cell, a tissue, an
embryo, or a fetus, is stained to saturation overnight in a solution of
0.1 M sodium cacodylate (pH 7.2), 1% glutaraldehyde, and 1% osmium
tetroxide, rocking at room temperature. The stained specimen is then
washed and dehydrated. For example, specimens are washed for 30 minutes
in 0.1 M sodium cacodylate buffer, and twice more for 30 minutes in
phosphate-buffered saline. Specimens are then incubated in a graded
series of ethanol concentrations to 100% ethanol prior to scanning.
Ethanol is an example of a medium that increases the apparent density
differences between the suspension medium and the stained tissue, thus
further increasing the level of contrast in images obtained from
specimens treated with such compositions.

[0097]The sensitivity and specificity of microCT-based analyses provides a
rapid and inexpensive method that enhances visualization and analysis of
complex global 3-dimensional organization. Unlike traditional histology,
which requires meticulous slicing and individual examination, the methods
of the present invention includes staining specimens with specific dyes
and scanning them with microscopic computed tomography (microCT), which
provides a high resolution image of the whole specimen without the need
for the slices required in other imaging modalities. The methods of the
present invention provide a digital visualization with the capability of
providing a number of measurements of various anatomical features of the
specimen. Such measurements include without limitation distance, area and
volume of such anatomical features.

[0098]Although the following section provides a description of embodiments
in terms of microCT imaging, it will be appreciated that these methods
can be adapted to other imaging technologies using methods known in the
art.

[0099]In specific embodiments, specimens prepared according to methods
known in the art and described herein are scanned in an X-ray computed
tomography scanner to provide microCT images of the specimens. Virtual
histology imaging methods are described in International Publication No.
WO/2007/089641, filed on Jan. 26, 2007 and U.S. application Ser. No.
11/575,057, filed Oct. 23, 2008, each of which is hereby incorporated by
reference in its entirety for all purposes and in particular for all
teachings related to microCT virtual histology.

[0100]A microCT image is generated, for example, using a commercially
available scanner, such as an eXplore Locus SP microCT specimen scanner
(GE Healthcare, London, Ontario) or the eXplore Locus RS small animal
microCT scanner (GE Healthcare, London, Ontario). More rapid volumetric
CT scans of specimens may be performed at lower resolution, such as at 27
micron3 isometric voxel resolution, while longer higher resolution
scans, such as 8 micron3 isometric voxel resolution, may also be
performed, depending on the desired cost, time constraints and resolution
required.

[0101]Parameters such as current, voltage, and exposure time are adjusted
as appropriate and are kept constant for images to be compared. For each
scan, a number of evenly spaced views may be averaged. The scans may be
filtered, for instance to avoid saturation of the detector, using
appropriate filters, such as 0.2 mm aluminum.

[0103]In an exemplary embodiment, specimen scans with resolution of 3
microns or better are obtained in less than 12 hours. For example,
isometric resolutions of 27 microns or 8 microns are achieved with scan
times of 2 hours or 12 hours. MicroCT-based virtual histology matches or
exceeds the tissue contrast achieved by more time- and cost intensive
magnetic resonance microscopy, while delivering more than 2-fold higher
resolution` up to 8 microns for microCT, (Jacobs, R. E., et al., Comput
Med Imaging Graph 23, 15-24 (1999), or in some cases up to 6 microns. For
increased throughput of these types of studies, multiple specimens are
optionally scanned simultaneously in the same field of view. For example,
at lower microCT resolutions (27 microns), multiple specimens can be
simultaneously scanned in approximately two hours with adequate quality
for post-imaging segmentation analysis allowing the recognition of gross
and subtle mutant phenotypes. For increased detail of abnormalities
suspected on the low-cost 27 micron scans, the same stained specimens can
later be scanned at 8 micron resolution for obtaining fine details such
as organ sub-compartments and fine tissue structures.

[0104]The computed tomography image of a specimen, such as an organ or
whole animal, may include an isosurface rendering so as to examine the
exterior of the specimen for anatomical or molecular differences compared
to other "control" specimens. In a further embodiment, the computed
tomography image of the specimen may include a virtual section of the
specimen.

[0105]Large numbers of images and associated data may be generated using
micro computed tomography to image specimens. Such virtual histology
datasets represent a valuable resource for investigating effects of
certain experimental procedures, such as for example, genetic
manipulation such as gene disruption or overexpression in vivo. However,
generated datasets relating to one mutation or other variable at a
particular stage of development or treatment may have further value when
compared to a second mutation/variable or at a second stage. In order to
facilitate access and aid in generation of such comparative data, a
computer-based process for collecting, storing and retrieving micro
computed tomography images and/or image data is provided according to the
present invention. In one embodiment, such a process includes the steps
of generating a digital computed tomography image, electronically
transmitting the image and/or data to a centralized data storage location
associated with a computer, retrieving the image and/or data from the
storage location in response to a request and electronically displaying
or transmitting the image and/or data and/or analysis of the image and/or
data to a second location in response to the request.

[0106]A generated computed tomography image and/or data for generating
such an image may be stored electronically, in memory circuitry such as a
database, and/or on a computer readable storage medium. A generated
computed tomography image is communicated to a repository for such
images, a centralized image and/or image data storage location associated
with a computer. Thus, for example, three-dimensional reconstructions of
transgenic and wild-type mouse embryos are generated and images and/or
data for image generation is sent to a centralized storage location
associated with a computer. Such images and data for image generation may
be generated and communicated from multiple locations for centralized
storage.

[0107]Communication of generated images and/or image data is may be
conducted over a wired or wireless connection to a device or system
configured as a server or computer network accessible by multiple users
from multiple locations. The server or computer network may include any
type of computer device or devices such as a personal computer,
workstation or mainframe computer.

[0108]Processing and memory circuitry is included in the server or
computer network such that an image and/or image data may be communicated
to memory circuitry and stored. Further, the stored information may be
retrieved from the memory circuitry. Optionally included is a comparison
program executable by the circuitry to carry out a comparison of one
images or set of images with another set of images in order to
characterize differences between the images relating to anatomical and/or
molecular differences in specimens imaged. Such a comparison program may
be stored and executed on a server or computer network which also
includes the stored image and/or image data. A comparison program may
also be stored and executed by a separate device to which images and/or
image data retrieved from the memory circuitry of the server or computer
network are downloaded.

[0109]An image and/or data for generating an image may be retrieved from
the centralized storage location in response to a request. For example, a
user inputs information to a device having data input and output capacity
to communicate a request to retrieve an image and/or image data from the
server or computer network storage location. The image and/or data may be
displayed to the user and/or downloaded to the user's device. Further,
the retrieved image and/or data may be retrieved for analysis and results
of the analysis displayed or downloaded to the user.

[0110]In some embodiments, multiple images of different specimens or
multiple images taken at different times of the same specimen will be
compared to identify differences and similarities in anatomical features.
In such embodiments, methods can be used to ensure that the images are
co-registered to identify points in each image which correspond to points
in the other images. Registration of images is a fundamental task in
image processing used to match two or more pictures taken, for example,
at different times, from different sensors, or from different viewpoints.
Registration techniques are known in the art. (see, e.g., Brown., (1992),
ACM Computing Surveys, 24(4): 325-76), and are also described in U.S.
application Ser. No. 11/839,414, filed on Aug. 15, 2007, which is hereby
incorporated by reference in its entirety for all purposes and in
particular for all teachings related to image processing and comparing
multiple images to each other and to reference images.

[0112]The washed specimens were then fixed in 10% neutral buffered
formalin for 4-5 days, followed by another series of washes in PBS. Three
PBS washes were conducted--each for an hour with a solution change after
each wash.

[0113]The specimens were then stained for four days in a PTA solution (1%
in water) at room temperature. The PTA solution was exchanged for fresh
solution each day. Some specimens were incubated in a staining
composition containing a standard 1% PTA solution, while other specimens
were stained in a stain that contained 1% PTA, 0.8 mM CaCl2 and
1.25×PBS [PBS=NaCl 137 mM, KCl 2.7 mM, and phosphate buffer 10 mM
(Na2HPO4/KH2PO4 pH 7.4) on 10× dilution.] The
stained specimens were washed and subjected to a microCT scan.

[0114]The specimens were then stained in a 5% PTA solution for three days.
Again, the PTA solution was exchanged for fresh solution each day. After
staining, the specimens were washed in PBS three times for one hour for
each wash. The washed specimens were then again subjected to a microCT
scan.

Example 2

Preparing Specimens for Staining

[0115]In order to increase penetration of one or more stains in a
specimen, the specimen may be blanched and/or incisions can be made in
the specimens prior to staining.

[0116]When using whole animal specimens, for example E16 to P0 mice or
rats, the specimen can be blanched and/or incisions may be made to open
the thoracic pleura, abdominal peritoneum, and/or dura mater to further
enhance stain penetration after skin removal.

[0117]The procedure for blanching the specimen can include making a small
shallow "x" cut on the ventral and dorsal sides of the specimen. The
specimen is placed in boiling water for approximately 10 to 12 seconds
and then doused in ice water. A cotton tip swab or other implement can be
used to gently rub the epidermis/dermis off of the specimen.
Alternatively, the skin may be peeled from the specimen using fine
forceps under a dissecting microscope. In order to remove extraneous
membrane and tissue, the specimen may be further sealed in a container
containing a solution such as PBS and placed on a rocking shaker for two
to ten minutes. The treatment with PBS and the rocking shaker may be
repeated multiple times as needed.

[0118]In addition to blanching, incisions may be made in the specimen to
further enhance the penetration of the staining composition into tissues
of interest.

[0119]To open the thoracic pleura, a short supracostal incision can be
made with a scalpel above the 10th rib on the left lateral side of the
body. Since nerves and vessels run below each rib, making the incision
above the rib will less likely cause damage to a vessel and avoid
unwanted hemorrhages. Additionally, since the 10th rib is located
anterior-lateral to the gap between the lungs and the diaphragm, making
the incision above the 10th rib will be less likely to cause damage to
internal structures.

[0120]Using scissors with the tips up, the cut is extended along the top
edge of the 10th rib to approximately 2-4 mm in length without damaging
internal structures such as the lungs and heart.

[0121]The supracostal incision/cut is then repeated for the right lateral
side of the body. The cut is generally no deeper than 1 mm from the
surface in order to open only the thoracic pleura and not damage any
internal organs.

[0122]To open the peritoneum, a small vertical incision can be made with a
scalpel along the midline of the abdominal cavity approximately 1 mm
above the umbilicus. Using micro-scissors with the tips up, the incision
is extended to approximately 1.3 mm in length in the direction of the
xiphoid process, cutting only the abdominal peritoneum without damaging
any internal organs. The incision is generally less than 1.3 mm in length
to ensure that the cut is inferior to the liver, thereby making it less
likely that the liver is damaged. The cut is also generally no deeper
than 0.3 mm from the surface to prevent damage to the intestines.

[0123]To open the dura mater, a 2-3 mm long incision with a scalpel can be
made along the suture of the skull. The cut is generally no deeper than
0.5 mm from the surface in order to open the dura mater without damaging
other structures in the brain.

[0124]Once all incisions are completed, the specimen can be transferred to
a staining or fixing solution for further processing.

Example 3

Preparation and Imaging of an Intact Knee Joint of a Rat

[0125]A rat was euthanized using institutionally approved protocols and an
intact knee of a hindlimb was isolated by cutting through the midshaft of
the femur and the midshaft of the tibia. The fascia surrounding the knee
was trimmed without compromising the integrity of the intact joint (see
FIG. 7).

[0126]The isolated knee sample was fixed in 10% Neutral Buffered Formalin
for about seven days with agitation by gentle rocking.

[0127]The knee sample was then removed from the 10% Neutral Buffered
Formalin and placed in 1×PBS to wash the excess fixation medium
from specimen. The wash was repeated until all excess fixation medium was
removed.

[0128]The knee sample was then placed in about twenty volumes of Hexabrix.
The sample was placed on a rocker to allow the stain to penetrate the
sample thoroughly for about twenty-four hours.

[0129]The sample was then removed from the Hexabrix and gently blotted dry
using an absorbent material until all excess staining fluid was removed.

[0130]The sample was then placed in minimally attenuating material and
secured for scanning. A small amount of aqueous solution was placed
within the scanning stage to ensure that the specimen did not dehydrate,
but the sample was not allowed to contact the aqueous solution. The
platform scanning parameters were adjusted to effectively visualize
cartilage as described in U.S. provisional application 61,143,383, filed
Jan. 8, 2009, which is herein incorporated by reference in its entirety
for all purposes and in particular for all disclosure (including written
description, figures and examples) related to visualizing cartilage.
Exemplary resultant images are shown in FIG. 3.

[0131]Seg3D was used to create a label map associated with the regions of
interest (cartilage and bone) from the imaging data for each specimen.
During this process, each voxel associated with a region of interest was
assigned a specific value (e.g. 1 for cartilage, 2 for bone and 0 for
background) which was then used for volume measurements. Teem
(http://teem.sourceforge.net/) was then used to convert the label map and
imaging data into frames for the planar movies and SCIRun (SCl Institute)
was used to generate the frames for the rotating 3D movies.

[0132]The following calculations were performed to compute the volume
measurements of the cartilage: [0133]1. After the segmentation process,
the voxel count associated with each region of interest was obtained
(i.e. the number of voxels associated with the cartilage were counted
using Seg3D). [0134]2. The voxel count for each region of interest was
then multiplied by the voxel resolution cubed to obtain volume
measurements. [0135]3. Based on the scanning parameters, the image data
was collected at 10 μm isometric voxel resolution (10/1000 mm); the
number of cubic millimeters in each voxel thereby translates into
(10/1000)3. The voxel count was multiplied by (10/1000)3 to
obtain the final measurement in millimeters cubed.

[0136]Surface Area Measurements: Based on the above obtained volume
measurements, the number of voxels along the outside edge were counted to
determine the surface area of the cartilage.

[0137]Thickness Map Generation: The distance from the bone to the edge of
the cartilage was calculated by obtaining the total number of voxels in
each distance then converting to millimeters. An image was generated
which translate the distances into a color map to allow for viewing of
the thickness along the length of the cartilage.

[0138]It is understood that the examples and embodiments described herein
are for illustrative purposes only and that various modifications or
changes in light thereof will be suggested to persons skilled in the art
and are to be included within the spirit and purview of this application
and scope of the appended claims. All publications, patents, and patent
applications cited herein are herein incorporated by reference in their
entirety for all purposes.